Preventive programs have been offered for the most differing cancers since the middle of the fifties. For cervical cancer an established population wide screening program exists in various developed countries. However similar screening programs are applicable for other cancer entities and the respective precursor stages such as e.g. cancers of the urinary system, of the respiratory tract and other. In the following cervical cancer is used as an example to highlight the drawbacks of the present preventive scenario. However the facts are mutandis mutatis applicable to other preventive programs for any cancer entity.
Regarding cervical intraepithelial neoplasia and cervical glandular lesions, the preventive programs are based mainly on the morphological and cytological examination of cytosmears of the cervix uteri, what is called the Pap test, which is made on the basis of gynecological routine examinations at regular intervals in women from the 20th year on. By means of the morphology of the cells, the smears are divided into various intensity degrees of dysplastic cellular changes. According to Pap I-V, these intensity degrees are referred to as normal, mild dysplasia, fairly serious dysplasia, serious dysplasia and invasive carcinoma, respectively. If the Pap test leads to a striking result, a small biopsy will be taken and subjected to a histopathologic examination, by which the kind and intensity of the dysplasia are determined and classified as cervical intraepithelial neoplasia (CIN1-3).
In spite of all preventive programs, cervical cancer that lead to 400,000 new cases per year is the second most frequent neoplastic disorder in women. This is inter alia due to the fact that up to 30% of the results of individual Pap test are false-negative.
In conventional screening for cervical intraepithelial neoplasia, swabs are used for detection of neoplastic lesions of the cervix uteri. In the screening procedure, different kinds of lesions have to be distinguished. Causes for lesions may for example be inflammations (due to infectious agents or physical or chemical damage) or neoplastic disorders. In morphological examinations the lesions of different characteristics are sophisticated to distinguish. Thus, for examination of cervical swabs and smears cytologists and pathologists have to be especially trained, and even experienced examiners have a high inter- and intra-observer variance in the assessment of a diagnosis based on cytological specimens. In general, the result of the examination is based upon the subjective interpretation of diagnostic criteria by the examining pathologist/cytologist. As a result, the rate of false positive and false negative results in the screening tests remains unsatisfying high.
However, the reproducibility of the examination results may be enhanced by the use of supporting molecular tools. Yet the problem with the preservation and preparation of the samples may not be overcome by just additionally using molecular markers. One further complication when performing cytological or histological examinations for screening purposes and especially when applying methods for the detection of molecular markers originates from strict precautions in preserving the samples from causing artefacts or improper results.
This is in part due to the instability of the cell-based morphological information and in part to the instability of the molecular markers to be detected during the tests. If the samples are not prepared, transported or stored in an appropriate manner, the cell-based information, or even the molecular information may be lost, or may be altered. So the diagnosis may be impossible, or may be prone to artefacts. For example, the interpretation of biopsies or cytological preparations is frequently made difficult or impossible by damaged (physically or bio/chemically) cells. Furthermore regarding tissue samples or biopsies, the preservation of molecular constituents of the samples, which are subject to a rapid turnover, is sophisticated due to the time passing by until penetration of the total sample by appropriate preservatives.
Although the above is shown using cervical cancer as an example the overall background also applies to preventive programs of neoplastic disorders in general as the situation for other cancer entities is very much the same. Generally the morphologically supported diagnostic methods performed routinely in the art show two major disadvantages. Firstly, the methods are highly dependent on individual perception of the examiners. Secondly, the morphological information is quite sensitive to decay processes and thus to production of artefacts after preparation of the samples. Both aspects contribute to improper reproducibility of the results.
Therefore, it is the object of the present invention to provide a method by which neoplastic disorders such as cancers and their precursor stages can be diagnosed early and reliably. In addition, a differentiation should be possible by this method with respect to benign inflammatory or metaplastic changes from neoplastic disorders such as dysplastic lesions and precancers. Moreover, the present invention provides methods for the detection of cancers on a biochemical basis from solubilized samples. The samples may be of any kind including cells in a cell preservation solution as is used for Liquid based cytology methods.
The inventors insight that use of LBC samples as a source of sample material for the development of diagnostic test kits for the biochemical non-cell based assessment of diagnosis of medically relevant conditions is another aspect of the present invention. In the art LBC samples are used for development of cell based assay formats. Lysis of the samples in a way as disclosed herein however enables inventors to base the development of the biochemical kits on sample material which is suited to provide information on the patients disease status from other diagnostic procedures on the same sample material.
A method for detection of HPV nucleic acids from LBC samples is disclosed by Digene Corp. This method uses LBC samples as basis for the analysis. Detection of the HPV nucleic acids is performed after lysis of the cells contained in the LBC samples. In this method no normalization of the amount of the LBC sample to be employed in the biochemical non-cell based detection of the HPV nucleic acid, is performed with respect to information obtained from the cytological specimen prepared out of the same LBC sample. The method disclosed by Digene is therefore restricted to mere qualitative measurements. Any biochemical non-cell based quantitative or even semiquantitative method needs information on the composition of the samples obtainable either from biochemical markers or from the microscopic or flow cytometric analysis of the sample. In the present invention the use of LBC samples for the assessment of diagnosis or for development of kits and in-vitro diagnostic devices enables for an accurate and comparable way to provide cytological information for the biochemical non-cell based testing. The employment of biochemical normalization with respect to markers indicative for the presence or absence of cells or cell types is omissible. The advantage of using LBC samples in this respect is that the cytologically cell based information is directly related to the homogeneous LBC specimen and thus provides valuable accurate information for use in the evaluation of the biochemical non-cell based test results.
A method for detection of molecular markers on the protein or nucleic acid level from solubilized specimens on the other hand is disclosed in various publications. However no link to the use of LBC samples as a source of the sample specimen in made in this respect. Generally LBC methods are applied in the art to enable for improved morphological evaluation of cytology specimens. The field of application of the LBC samples is therefore indicated only for cytology. Based on the disclosure in the prior art preparation of an LBC sample for subsequent solubilization of the sample for biochemical testing is not disclosed. Moreover the disclosure as to the advantages of LBC procedures teach away from application of LBC samples in any method that is not founded on cellular morphological evaluation of the specimens. According to the inventors findings the use of LBC samples as a source for biochemical non-cell based determination of protein levels in solubilized specimens provides the advantage that the results may be directly compared to a cytological specimen. The protein based biochemical analysis in this respect may serve as a e.g. pre-testing or to provide further information or even to confirm a cytologically equivocal result. In further embodiments the information obtained from the biochemical non-cell based testing may be for the design of the cytological procedures to be applied.
The development method disclosed herein is therefore of great value for achieving effective and reliable kits and in-vitro diagnostic devices. The method for development of kits and in-vitro diagnostic devices as disclosed herein achieves comparability of the results generated by biochemical non-cell based analysis with the cytologically assessed results by means of a normalization. This normalization of the sample for application in the biochemical test format is performed with respect to information on the LBC sample obtainable from the cytological specimen prepared from the LBC sample. Such information comprises e.g. cellularity of the LBC sample, information with respect to volume of the LBC sample, information with respect to mass of the LBC sample or with respect to parameters accessible only via the generation of a thin-layer specimen out of the LBC sample. In this respect the inventors provide by the methods as claimed herein a reliable method for development of kits and in-vitro diagnostic devices on the basis of LBC samples.